Production of Protease Enzyme from Microorganisms

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1. WELCOME

2. Tittle “Production of Protease Enzyme from Microorganisms”:
MSC Biotechnology II
Chaitanya Patil
Saniket Patil














Abstract
Protease are a group of hydrolytic enzyme that catalyze the breakdown of protein into Amino
Acids by cleaving the peptide bond. They have significant industrial applications in sectors
such as food processing, detergents, pharmaceuticals, leather, and biotechnology.

3. Microorganisms, including bacteria, fungi, and actinomycetes, are considered the most
promising sources of proteases due to their rapid growth, ease of cultivation, and ability to
produce large quantities of enzymes under controlled conditions. This study focuses on the
isolation, screening, and production of protease enzymes from selected microorganisms.
Microbial strains are cultivated on specific protein-rich media to induce enzyme secretion,
followed by optimization of parameters such as pH, temperature, incubation time, and
substrate concentration to maximize protease yield. The crude enzyme is extracted, partially
purified, and characterized for its activity. The results are expected to demonstrate the
efficiency of microbial sources in producing cost-effective and eco-friendly proteases suitable
for various industrial applications. This research contributes to sustainable enzyme
production by utilizing microbial fermentation as an alternative to chemical processes.

Introduction

Microorganisms are widely used in biotechnology for the large-scale production of enzymes.
Among them, Bacillus species are the most common producers of extracellular proteases,
particularly alkaline proteases. These enzymes are vital due to their wide industrial
applications in detergents, pharmaceuticals, leather, food, agriculture, and waste treatment.
Proteases play essential roles in cellular processes and occupy nearly 60% of the global
enzyme market. They are highly stable, active under alkaline conditions, and can be
genetically modified to enhance production. Factors like pH, temperature, and nutrient
availability influence their yield. Due to their rapid growth and easy cultivation, Bacillus
subtilis is the preferred microorganism for protease production.






Aim and objective:
The main objective of this research to isolate protease
enzyme producing by microorganism from soil.

4. -collection of soil sample from subcampus osmanabad,
-Isolation of microorganism from soil sample by serial
dilution.
-Screening of protease producers.
-Production of protease using different media.
-Purification of protease enzyme -Assay of protease
enzyme











Methodology of Protease Production & Assay
1. Collection and Isolation of Samples
⬇️ Soil samples collected → Serial dilution (10⁻³ to 10⁻⁸) → Spread on nutrient agar →
Incubate 24h at 37°C → Colonies grown

5. 2. Screening of Protease Producers
⬇️ Colonies picked → Streaked on skim milk agar → Incubate 24h at 37°C → Zone of
hydrolysis observed → Positive colonies subcultured & stored

3. Production of Protease Using Different Media
⬇️ Media used: Nutrient broth, Luria bertani broth, Lactose broth, Soybean casein broth,
Potato dextrose broth (PDA)
⬇️ Inoculate colonies into 100 ml broth → Incubate 72h at 37°C → Centrifuge at 5000 rpm,
30 min → Collect supernatant

4. Separation & Purification of Protease
⬇️ Add ammonium sulfate (75% saturation) → Precipitate protein (shaking + magnetic stirrer)
⬇️ Centrifuge at 5000 rpm, 30 min → Dissolve in phosphate buffer (pH 7) → Dialyze →
Store crude enzyme

5. Quantitative Assay of Protease
⬇️ Reaction mixture: 0.5 ml gelatin + 1 ml culture filtrate
⬇️ Incubate 37°C, 10 min → Add 5 ml trichloroacetic acid → Stop reaction
⬇️ Centrifuge at 5000 rpm, 30 min → Collect supernatant
⬇️ Add 5 ml alkaline solution + 1 ml Folin phenol reagent → Incubate 30 min, room temp
⬇️ Measure protease activity (colorimeter)

6. THANK YOU